During the previous funding cycle we identified five monoclonal antibodies (MAbs) against the P1 surface adhesin of Streptococcus mutans that redirect the humoral immune response in mice in terms of quantity, specificity and isotype of elicited antibodies when administered with the antigen as an immune complex. This strategy has implications for vaccine design in that protective immunity is not necessarily directed at immunodomiant epitopes of pathogens and could well be improved by shifting a response toward subdominant epitopes. Results varied depending on the anti-P1 MAb tested. Two MAbs resulted in formation of antibodies more inhibitory of S. mutans adherence in vitro and two others of less inhibitory antibodies. Epitopes recognized by a panel of anti-P1 MAbs were characterized and include complex discontinuous determinants. Polyclonal antibodies from immunized mice also recognize conformational epitopes, some of which appear more biologically relevant than others. Significant positive correlations were demonstrated between the ability of anti-P1 MAbs to inhibit S. mutans adherence and IgG2a and IgG2b isotypes suggesting the relevance of cytokines involved in class switching in the development of beneficial responses. This will be explored as part of the current proposal. Binding of an anti-P1 MAb to cell-associated P1 altered its susceptibility to numerous proteases in vitro indicating an influence on protein structure and suggesting exposure of cryptic epitopes and changes in antigen processing and presentation. Studies to address this potential mechanism of immunomodulation will also be undertaken. We will continue to characterize MAb-mediated changes in humoral immune responses in immunized mice to identify correlates of protection against S. mutans adherence in vitro and colonization and cariogenicity in vivo. Immunoassays of samples from immunized mice will utilize combinations of P1 segments, or deletion constructs, known to reconstitute or destroy conformational epitopes. The anti-P1 response against conformational determinants has not been studied to date in vaccinated animals or naturally sensitized humans. Immunomodulation by anti-P1 MAbs represents a strategy to significantly improve the humoral response against S. mutans and provides a tool to dissect beneficial and non-beneficial antibodies against this widely studied candidate vaccine antigen. The characterization of epitopes recognized by anti-P1 MAbs that improve or decrease the beneficial response will provide insight into the structure of P1 required to elicit optimal protective immunity and is expected to shed light on ways to modify the protein itself to improve its protective immunogenicity without the need to immunize with immune complexes. Beyond the impact of these studies on development of a therapeutic approach against human dental caries, the well-characterized P1 antigen and MAbs against it represent an excellent model system to understand the consequences and underlying molecular mechanisms of immunomodulation and would be of general interest for any active or passive immunization approach.